PROJECT SUMMARY Prion diseases are infectious neurodegenerative disorders caused by PrPSc, a misfolded and aggregated isoform of the cellular prion protein, PrPC. During late disease stages, PrPSc spreads through the CNS, causing neurodegeneration, gliosis, and ultimately death. Earlier studies suggest that prions spread through neuroanatomically connected brain regions, however the mechanisms of prion transport remain poorly defined. Here we propose to investigate the role of exosomes in the intercellular spread of prions. Exosomes are extracellular nanovesicles that originate from the endosomal pathway and are important for intercellular communication, transporting proteins and mRNA to nearby cells. Exosomes harbor infectious prions isolated immortalized cell lines and mice, and these exosomes efficiently cause prion disease in mice. I hypothesize that prions exploit the exosome pathway as the primary mechanism of prion spread into and within the CNS. I will address this hypothesis in three aims using both in vitro and in vivo models. In the first aim, I will define the size of prion aggregates packaged into exosomes using recombinant PrP fibrils and a diverse population of brain-derived prion strains, as well as measure prions in exosomes isolated from prion-infected cells and prion- infected mouse brain. In the second aim, I will manipulate the early vesicular pathway (Hrs in ESCRT-0) in vitro and determine the effect on prion spread between cells using primary neurons, astrocytes, and immortalized neurons in transwell systems. In the third aim, I will manipulate Hrs in the exosome pathway in a cell-specific manner in vivo and investigate the impact on prion neuroinvasion and spread through the CNS. I expect that these studies will elucidate mechanisms of protein aggregate spread that will be critical for understanding protein misfolding diseases and developing novel therapeutic strategies to arrest disease progression.